Name: Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier
Uploaded: 2023-10-20T12:30:00
Description: Watch this Scientific Journal Video about Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier at JoVE.com

constructing the 3d human-stem-cell-derived in vitro blood brain barrier

源自人类多能干细胞的 3D 血脑屏障模型

The blood-brain barrier (BBB) is a key microvascular network separating the central nervous system (CNS) from the periphery to maintain an ideal environment for proper neuronal function. It has a critical role in regulating the influx and efflux of substances into the CNS by maintaining metabolic homeostasis1,2,3,4, clearing waste4,5,6, and protecting the brain from pathogens and toxins7,8.
The primary cell type of the BBB is the endothelial cell (EC). Endothelial cells, derived from the mesoderm lineage, form the walls of the vasculature1,9. Microvascular ECs form tight junctions with each other to greatly decrease the permeability of their membrane10,11,12,13,14 while expressing transporters to facilitate the movement of nutrients into and out of the CNS1,4,12,14. Microvascular ECs are encircled by pericytes (PCs)-mural cells that regulate microvascular function and homeostasis and are critical for regulating the permeability of the BBB to molecules and immune cells15,16,17. The astrocyte, a major glial cell type, is the final cell type comprising the BBB. Astrocyte end-feet wrap around the EC-PC vascular tubes while the cell bodies extend into the brain parenchyma, forming a connection between neurons and vasculature1. Distinct solute and substrate transporters are localized on astrocyte&#160;end-feet (e.g., aquaporin 4 [AQP-4]) that have a critical role in BBB function18,19,20,21.
The BBB is critical in maintaining proper brain health function, and dysfunction of the BBB has been reported in many neurological diseases, including Alzheimer&#39;s disease (AD)22,23,24,25, multiple sclerosis7,26,27,28, epilepsy29,30, and stroke31,32. It is increasingly recognized that cerebrovascular abnormalities play a central role in neurodegeneration, contributing to increased susceptibility to ischemic and hemorrhagic events. For example, more than 90% of AD patients have cerebral amyloid angiopathy (CAA), a condition characterized by the deposition of amyloid &#946; (A&#946;) along the cerebral vasculature. CAA increases BBB permeability and decreases BBB function, leaving the CNS vulnerable to ischemia, hemorrhagic events, and accelerated cognitive decline33.
We recently developed an in vitro model of the human BBB, derived from&#160;patient-induced pluripotent stem cells, which incorporates ECs, PCs, and astrocytes encapsulated in a 3D matrix (Figure 1A). The iBBB recapitulates physiologically relevant interactions, including vascular tube formation and localization of astrocyte end-feet with vasculature24. We applied the iBBB to model the susceptibility of CAA mediated by APOE4 (Figure 1B). This enabled us to identify the causal cellular and molecular mechanisms by which APOE4 promotes CAA, and leverage these insights to develop therapeutic strategies that reduce CAA pathology and improve learning and memory in vivo in APOE4 mice24. Here, we provide a detailed protocol and video tutorial for reconstructing the BBB from human iPSCs and modeling CAA in vitro.

作者聚焦：使用体外血脑屏障模型研究阿尔茨海默病的个性化方法 

体外血脑屏障重建，模拟和治疗神经系统疾病

Our goal is to determine the molecular and cellular mechanisms that mediate Alzheimer's disease pathogenesis to uncover therapeutic and diagnostic opportunities. Currently, the molecular pathways and mechanisms involved in the onset and development of Alzheimer's disease are poorly understood. This is due to mouse models and other in vitro Alzheimer's disease models failing to recapitulate unique human biology that is crucial to the development of the disease.We have developed an in vitro model of the blood-brain barrier that can be applied to study cerebrovascular pathologies associated with Alzheimer's disease, dementia, and other forms of neurodegeneration. This model has been employed to show that APOE4, the strongest risk factor for Alzheimer's disease, acts in part through a parasite-specific pathway to increase pathogenic amyloid burden Using induced pluripotent stem cells, we can construct human brain tissue in vitro that can be used to investigate disease pathology and drug screenings. Such an in vitro blood-brain barrier model enables personalized therapeutic and diagnostic approaches.Our 3D in vitro blood-brain barrier model demonstrates physiologically relevant interactions, including vascular tube formation and localization of astrocyte endfeet with vasculature. It is also capable of modeling disease-relevant phenotypes, including amyloid pathology. Finally, the use of patient-derived induced pluripotent stem cells allows the study of any desired genetic background.Using the in vitro blood-brain barrier model, we discovered pathways that mediate pathological cerebrovascular amyloid accumulation. This led us to explore therapeutic strategies that can reverse this pathology in aged APOE4 mice, which we are currently following up on.

Watch this Scientific Journal Video about Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier at JoVE.com

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier  

构建3D人干细胞来源的体外血脑屏障

使用解离的内皮细胞、寄生虫和星形胶质细胞组装人多能干细胞衍生的血脑屏障或 iBBB，将三种类型的细胞组合在一个锥形管中，同时包括比计算的细胞数量多 10% 以考虑移液错误。将细胞混合物以 300 g 旋转三分钟。吸出上清液，使细胞沉淀中含有约50μL的培养基。使用P200移液管，轻轻地将细胞沉淀重悬于残留培养基中，以产生单细胞浆液。将含有所得细胞浆液的试管放在冰上，并根据所需数量的 iBBB 向其添加足量的还原 GF 基底膜基质。均匀混合细胞，同时不引入任何气泡。将 50 μL 所得细胞基质混合物移液到 48 孔或 96 孔玻璃底培养皿的孔中，并将其均匀分布在整个培养皿底部。将培养皿在 37 摄氏度下孵育 30 至 40 分钟，以聚合基质，同时封装细胞。最后，向孔中加入 500 微升补充的星形胶质细胞培养基，并在培养物中维持 iBBB。对于 96 孔板，向孔中加入 100 至 150 μL 培养基，直到它们充分显影用于下游测定，这通常需要两周时间。正确形成的iBBB在明场显微镜下显示为凝固的单个半透明圆盘。24小时后，鉴定出均匀分布的单细胞。两周后，虽然难以定义，但可以看到更明显的结构。

Research

JoVE Journal

Neuroscience

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...